Assessing Milk Protein Stability Using ATR-FT-IR Spectroscopy

Scientists from the Teagasc Food Research Centre and University College Dublin, in Ireland led by Mark A. Fenelon, Head of Food Programme at Teagasc, examined how milk proteins respond to heat. Published in the International Journal of Dairy Technology, the study demonstrated how attenuated total reflectance (ATR)-Fourier transform infrared (FT-IR) spectroscopy can be used to monitor heat-induced changes in milk proteins (1).

Whey protein scoop. Sports nutrition. | Image Credit: © Nick Starichenko - stock.adobe.com

Monitoring milk proteins is important in the dairy production process to ensure the dairy products are of high quality. There are different types of milk proteins. One type is milk proteins is whey proteins, which come in three main types: whey protein concentrate (WPC), whey protein isolate (WPI), or hydrolyzed whey protein (2). Another type is caseins, which can be comprised of sodium, calcium, and other elements (2). While whey proteins and caseins have some differences between the two, both undergo structural changes when exposed to high temperatures, affecting their functionality, texture, and shelf life (1, 2). As a result, it is important for dairy product manufacturers to understand these changes, because knowing this information can help them improve heat stability in dairy products, reduce waste, and ensure consistent quality in processed milk (1).

In this study, the research team tracked protein structural changes in real-time and evaluate the correlation between milk protein changes and heat stability using ATR-FT-IR spectroscopy.

As part of the experimental procedure, the team collected raw skim milk directly from a bulk tank and subjected it to rigorous analysis. The team conducted five replicate trials between February and May 2022, during which milk composition—including fat, total protein, lactose, and total solids—was measured using Fourier transform mid-infrared (FT-MIR) spectroscopy (1). After centrifugation to remove fat, the skim milk samples were adjusted to three different pH levels: 6.2, 6.8 (native pH), and 7.0 (1).

The results demonstrated that milk samples at pH 6.2 exhibited the fastest rate of protein denaturation, as indicated by shifts in the second derivative of the amide I region of the FT-IR spectra (1). The spectral changes at 1624 cm⁻¹, associated with β-sheet structures in β-lactoglobulin (β-Lg), confirmed heat-induced denaturation (1).

In contrast to the above results, milk samples at pH 6.8 and pH 7.0 structurally changed much more slowly, which means that there was greater heat stability. The researchers also observed interactions between casein and whey proteins in the spectral region between 1655 cm⁻¹ and 1672 cm⁻¹, further demonstrating the role of pH in influencing protein behavior under heat stress (1).

Another critical aspect of the study was the role of colloidal calcium phosphate (CCP) in protein stability. The researchers found that spectral peaks at 1072 cm⁻¹, associated with CCP, increased as temperature and pH rose (1). This observation is indicative of more extensive CCP transformations between colloidal and serum phases occur at higher temperatures and pH levels, which influenced protein aggregation (1).

Another key observation was that changes in CCP didn’t exist in the whey samples. This observation suggests that casein micelles play a key role in this process (1). As a result, the conclusion was that CCP-protein interactions play a role in determining milk’s heat stability (1).

The study demonstrates that ATR-FTIR spectroscopy is a powerful tool for assessing milk’s thermal stability in real time. By providing direct, in situ measurements of protein structural changes and CCP transformations, this technique could help dairy manufacturers optimize processing conditions, enhance product quality, and minimize waste (1). This method could be extended beyond milk to dairy concentrates and other formulations, providing a valuable analytical approach for improving heat stability in a wide range of dairy products (1).

References

1. Hayes, E.; Kelly, L.; Aydogdu, T.; et al. Real-time Measurement of Heat Stability of Skim Milk Using Attenuated Total Reflectance (ATR)-FTIR Spectroscopy. Int. J. Dairy Technol. 2025, 78 (1), e13156. DOI: 10.1111/1471-0307.13156
2. Agropur, Proteins From Milk: Types and Characteristics. Agropur.com. Available at: https://www.agropur.com/us/news/proteins-from-milk (accessed 2025-03-17).